Periodic Reporting for period 2 - FuncDis3D (Deconstructing gene regulation through functional dissection of the 3D genome)
Reporting period: 2022-03-01 to 2023-08-31
Every cell in our body that contains a nucleus stores 2 meters of DNA in a volume that is only 1/100th of millimeter across (or 1/10th of the width of human hair). This is achieved through the non-random folding of the DNA. Correct folding of DNA is crucial for processes such as DNA replication, DNA repair and gene transcription. Failures in these processes can lead to diseases such as cancer or developmental abnormalities (i.e. congenital defects). Therefore a proper understanding behind the mechanisms that drive the folding of DNA inside the nucleus is important to understand how regulatory processes can go wrong.
A large group of proteins is crucial to the correct folding of the DNA inside the nucleus. However, many of these proteins are essential for the survival of the cell. Therefore, standard loss-of-function analyses (i.e. knock-out or knock-down) does not work for these proteins. Studying genome organization is further complicated by the fact that the genome organization is highly dynamic. To circumvent this problem, we have implemented so-called acute protein depletion methods. Using these methods we are able to achieve near-complete depletion of a specific protein with sub-hour time resolution. This allows us to chart rapid differences in the organization of the genome.
We profile different (genome-wide) molecular characteristics (i.e. transcription, 3D genome organization, chromatin binding and accessible chromatin) and use sophisticated computational analyses to identify associations between the different molecular components. This enables us to predict cause and consequence in the changes that we observe.
In the final part of the project we aim to determine how regulators of the 3D genome affect differentiation. One of the reasons for this is that many developmentally important genes are regulated by sequences that are far away on the chromosome template, but interact with each other in the space of the nucleus. We want to understand how misregulation of these genes is affected during (in vitro) development.
A large group of proteins is crucial to the correct folding of the DNA inside the nucleus. However, many of these proteins are essential for the survival of the cell. Therefore, standard loss-of-function analyses (i.e. knock-out or knock-down) does not work for these proteins. Studying genome organization is further complicated by the fact that the genome organization is highly dynamic. To circumvent this problem, we have implemented so-called acute protein depletion methods. Using these methods we are able to achieve near-complete depletion of a specific protein with sub-hour time resolution. This allows us to chart rapid differences in the organization of the genome.
We profile different (genome-wide) molecular characteristics (i.e. transcription, 3D genome organization, chromatin binding and accessible chromatin) and use sophisticated computational analyses to identify associations between the different molecular components. This enables us to predict cause and consequence in the changes that we observe.
In the final part of the project we aim to determine how regulators of the 3D genome affect differentiation. One of the reasons for this is that many developmentally important genes are regulated by sequences that are far away on the chromosome template, but interact with each other in the space of the nucleus. We want to understand how misregulation of these genes is affected during (in vitro) development.
We have generated or acquired through collaborations acute depletion lines for a number of proteins that are known or thought to be involved in genome organization. For instance: RAD21, WAPL, CTCF, YY1, ZFP143, SMARCB1, SOX2, NANOG and NIPBL. In addition, we have made combination of proteins such as CTCF and WAPL, furthermore we have combined two different depletion systems which allows us to deplete proteins independently, which allows us to dissect cause and effect. For instance, we have generated a degron line in which CTCF and WAPL are tagged with one depletion system (auxin inducible degron) and RAD21 with another (dTAG system). We have also generated a similar degron line with CTCF/WAPL and SETD2.
Our analyses have found that SETD2 is a novel looping factor that brings actively transcribed genes together. The precise mechanism is not yet fully clear, but we are currently trying to determine whether it is the enzymatic activity of the protein or the protein itself that is important for the loop formation.
In addition, to identifying novel proteins that are involved in looping, we have compelling evidence that the transcriptional activator ZFP143 is not a looping factor. Although, genomics studied have shown that ZFP143 is thought to be associated with chromatin loops, acute depletion of this factor does not affect the 3D genome. Interestingly, we find that ZFP143 is important for the transcriptional activation of a large number of genes. Among these are the ribosomal proteins. We are currently investigating the downstream consequences of ZFP143 inactivation on cell proliferation and translation.
Our analyses have found that SETD2 is a novel looping factor that brings actively transcribed genes together. The precise mechanism is not yet fully clear, but we are currently trying to determine whether it is the enzymatic activity of the protein or the protein itself that is important for the loop formation.
In addition, to identifying novel proteins that are involved in looping, we have compelling evidence that the transcriptional activator ZFP143 is not a looping factor. Although, genomics studied have shown that ZFP143 is thought to be associated with chromatin loops, acute depletion of this factor does not affect the 3D genome. Interestingly, we find that ZFP143 is important for the transcriptional activation of a large number of genes. Among these are the ribosomal proteins. We are currently investigating the downstream consequences of ZFP143 inactivation on cell proliferation and translation.
The double depletion lines (using two different depletion systems) are an innovation that allows us to explore cause-and-effect relationships on very short timescales. We have found that certain features of the 3D genome are driven by the cohesin complex. In addition, we have identified a novel type of chromatin loops that is driven by a histone modifying enzyme. We are now investigating the mechanism of formation of these loops and their function.
To explore the role of the 3D genome in development, we have implemented an in vitro embryo model called gastruloids. In this model early lineage specification events are captured. By combining this with our acute depletion lines we can deplete proteins at specific timepoints in development and determine the effect on cell type determination and morphology. We have identified a number of 3D genome regulators that affect the morphology of gastruloids. We are currently systematically exploring what factors are responsible for disrupting gastruloid morphology.
The group is split roughly into 50/50 in experimental researchers and computational researchers. We are constantly developing novel analysis tools for 3D genome analysis and single cell genomics data. One example of a computational tool that we have developed is GENOVA. This R package is freely available on GitHub and combines many standard analysis tools for Hi-C data analysis. It is written specifically with novice users in mind and allows experimental researchers to analyze their own Hi-C data. From the feature requests we get, it is clear that the package is used extensively. We will provide other software that we develop to the community as well.
To explore the role of the 3D genome in development, we have implemented an in vitro embryo model called gastruloids. In this model early lineage specification events are captured. By combining this with our acute depletion lines we can deplete proteins at specific timepoints in development and determine the effect on cell type determination and morphology. We have identified a number of 3D genome regulators that affect the morphology of gastruloids. We are currently systematically exploring what factors are responsible for disrupting gastruloid morphology.
The group is split roughly into 50/50 in experimental researchers and computational researchers. We are constantly developing novel analysis tools for 3D genome analysis and single cell genomics data. One example of a computational tool that we have developed is GENOVA. This R package is freely available on GitHub and combines many standard analysis tools for Hi-C data analysis. It is written specifically with novice users in mind and allows experimental researchers to analyze their own Hi-C data. From the feature requests we get, it is clear that the package is used extensively. We will provide other software that we develop to the community as well.